The efficiency of mosquito infections by arthropod-borne viruses (arboviruses) can be an important determinant of disease emergence. An example is Venezuelan equine encephalitis virus (VEEV), an important, reemerging alphavirus pathogen of humans and equines in the New World, and also a potent biological weapon. Our recent work has demonstrated that enzootic, sylvatic, rodent-borne, subtype ID VEEV strains periodically adapt for efficient equine replication and high titer viremia induction to generate epidemic strains and amplification cycles that spillover to infect humans. These adaptation events are mediated by mutations that alter amino acids in the E2 envelope glycoprotein, an important component of spikes on the virion surface. In addition, the E2 genes of most subtype IAB and IC epidemic strains also enhance their ability to infect the epidemic mosquito vector, Aedes (Ochlerotatus) taeniorhynchus, a salt marsh species that has been implicated in transmission during most major VEE outbreaks ranging from northern South America to Texas. Either adaptation or the pre-existing ability of VEEV to utilize this vector (possibly due to selection for equine amplification) may therefore be a major determinant of epidemic transmission and outbreak magnitude. This virus-vector interaction is also an ideal model system to advance understanding of natural alphavirus-mosquito interactions because it has been thoroughly evaluated using a variety of viral strains and mosquito populations. We will further characterize infection of Ae. taeniorhynchus by both enzootic and epidemic VEEV strains in order to understand more completely the mechanisms of viral adaptation to this vector. The enhanced epidemic VEEV strain infection, dissemination in and/or transmission by Ae. taeniorhynchus will be examined using a unique set of fluorescent protein- expressing enzootic and epidemic VEEV replicon particles and viruses. Combined with traditional and molecular genetic methods, these tools will be used to examine infection of the mosquito midgut, believed to be the portal of entry, as well as downstream target tissues and organs including the salivary glands. These viruses and replicon particles will also be used to characterize the barriers to infection and dissemination that are overcome by epidemic VEEV strains for efficient transmission, and reverse genetic approaches will identify the determinants of the efficient infection VEEV phenotype. The results of this project will enhance understanding of natural interactions between alphaviruses and mosquito vectors, a research topic that has received little attention in recent years. It will also add to our understanding of the mechanisms of epidemic VEEV emergence and provide new viral and host targets for the development of safer vaccines and novel strategies to interrupt arbovirus transmission.This project will examine the process of infection of mosquito vectors by Venezuelan equine encephalitis virus (VEEV), an important emerging virus that continues to threaten the Americas including the U.S. The mechanisms and genetic determinants of mosquito infection will be determined to explain why certain mosquitoes are efficient vectors of some but not other VEEV strains. The results will add to our understanding of the mechanisms of epidemic VEEV emergence and provide new viral and host targets for the development of safer vaccines and novel strategies to interrupt transmission.